Crystalline forms of a 3-carboxypropyl-aminotetralin compound

ABSTRACT

The invention provides crystalline solid forms of (S)-4-((2S,3S)-7-carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydronaphthalen-2-ylamino)-2-cyclohexylmethyl-butyric acid. The invention also provides pharmaceutical compositions comprising such crystalline solid forms, methods of using such crystalline solid forms to treat diseases associated with mu opioid receptor activity, and processes useful for preparing such crystalline solid forms.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. Ser. No.13/663,083, filed Oct. 29, 2012, now U.S. Pat. No. 8,486,958 B2, whichis a divisional application of U.S. Ser. No. 13/330,358, filed Dec. 19,2011, now U.S. Pat. No. 8,324,236 B2, which is a divisional applicationof U.S. Ser. No. 12/633,944, filed Dec. 9, 2009, now U.S. Pat. No.8,101,794 B2, which claims the benefit of U.S. Provisional ApplicationNo. 61/121,254, filed on Dec. 10, 2008, the disclosures of which areincorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention is directed to crystalline forms of a3-carboxypropyl-aminotetralin compound which are useful as mu opioidreceptor antagonists. The invention is also directed to pharmaceuticalcompositions comprising such crystalline compounds, methods of usingsuch compounds for treating or ameliorating medical conditions mediatedby mu opioid receptor activity, and processes useful for preparing suchcompounds.

2. State of the Art

Mu opioid receptor antagonists are expected to be useful for treating orameliorating medical conditions mediated by mu opioid receptor activity.In particular, peripherally selective mu opioid receptor antagonists areexpected to be useful for treating conditions such as opioid-inducedbowel dysfunction and postoperative ileus. Commonly-assigned U.S.Provisional Application Nos. 61/007,220, filed Dec. 11, 2007, and61/049,219, filed Apr. 30, 2008 and U.S. application Ser. No.12/331,659, filed Dec. 10, 2008, disclose 3-carboxypropyl-aminotetralincompounds. In particular, the compound(S)-4-((2S,3S)-7-carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydronaphthalen-2-ylamino)-2-cyclohexylmethyl-butyricacid (compound 1):

is specifically disclosed in these applications as demonstrating muopioid receptor antagonist activity.

To effectively use this compound as a therapeutic agent, it would bedesirable to have a solid-state form that can be readily manufacturedand that has acceptable chemical and physical stability. For example, itwould be highly desirable to have a physical form that is thermallystable, for example at temperatures exceeding about 160° C. or about180° C., and is not deliquescent, thereby facilitating processing andstorage of the material. Crystalline solids are generally preferred overamorphous forms, for enhancing purity and stability of the manufacturedproduct.

No crystalline forms of compound 1 have previously been reported.Accordingly, a need exists for a stable, crystalline form of compound 1that is not deliquescent, and exhibits favorable thermal stability.

SUMMARY OF THE INVENTION

The present invention provides two distinct crystalline solid forms of(S)-4-((2S,3S)-7-carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydronaphthalen-2-ylamino)-2-cyclohexylmethyl-butyricacid (1) and a crystalline hydrochloride salt of compound 1.

Surprisingly, one form of crystalline compound 1 has been found toexhibit no significant thermal events below a temperature of about 162°C. and to exhibit a weight change of less than about 2.5% when exposedto a range of relative humidity between about 2% and about 90% at roomtemperature. Furthermore, neither crystalline compound 1 nor thecrystalline hydrochloride salt of compound 1 is deliquescent whenexposed to up to about 90% relative humidity at room temperature.

Among other uses, the crystalline solid forms of the invention areexpected to be useful for preparing pharmaceutical compositions fortreating or ameliorating medical conditions mediated by mu opioidreceptor activity. Accordingly, in another of its composition aspects,the invention provides a pharmaceutical composition comprising apharmaceutically-acceptable carrier and crystalline(S)-4-((2S,3S)-7-carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydronaphthalen-2-ylamino)-2-cyclohexylmethyl-butyricacid or the present crystalline hydrochloride salt.

The invention also provides a method of treating or ameliorating adisease or condition ameliorated by treatment with a mu opioid receptorantagonist, e.g. a disorder of reduced motility of the gastrointestinaltract, the method comprising administering to the mammal, atherapeutically effective amount of crystalline(S)-4-((2S,3S)-7-carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydronaphthalen-2-ylamino)-2-cyclohexylmethyl-butyricacid (1) or a crystalline hydrochloride salt of compound 1.

The invention further provides a method of treating opioid-induced boweldysfunction or post-operative ileus, the method comprising administeringto the mammal, a therapeutically effective amount of crystalline(S)-4-((2S,3S)-7-carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydronaphthalen-2-ylamino)-2-cyclohexylmethyl-butyricacid (1) or a crystalline hydrochloride salt of compound 1.

In another method aspect, the invention provides a process for preparinga crystalline compound 1 in Form I, the process comprising dispersing(5)-4-((2S,3S)-7-carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydronaphthalen-2-ylamino)-2-cyclohexylmethyl-butyricacid (1) in a polar diluent comprising between about 3% and about 20%water to form a crystallization process mixture; holding the processmixture for at least about 12 hours; and isolating the resultingcrystals from the process mixture.

In yet another method aspect, the invention provides a process forpreparing crystalline compound 1 in Form I, the process comprisingdeprotecting the protected intermediate,(S)-4-((2S,3S)-7-carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydronaphthalen-2-ylamino)-2-cyclohexylmethyl-butyricacid benzyl ester by catalytic hydrogenolysis in the presence of a polardiluent comprising between about 10% and about 20% water to formcrystalline compound 1.

In related composition aspects, the invention provides(S)-4-(2S,3S)-7-carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydronaphthalen-2-ylamino)-2-cyclohexylmethyl-butyricacid benzyl ester and the bisulfate adduct, sodium;(S)-3-benzyloxycarbonyl-4-cyclohexyl-1-hydroxy-butane-1-sulfonate, whichis useful for the preparation of the above protected precursor tocompound 1.

The invention also provides crystalline solid forms of the invention asdescribed herein for use in therapy or as a medicament, as well as theuse of a crystalline solid form of the invention in the manufacture of amedicament, especially for the manufacture of a medicament for treatinga disorder associated with mu opioid receptor activity in a mammal.

BRIEF DESCRIPTION OF THE DRAWINGS

Various aspects of the present invention are illustrated by reference tothe accompanying drawings.

FIG. 1 shows an x-ray powder diffraction (XRPD) pattern of crystallineForm I(S)-4-((2S,3S)-7-carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydronaphthalen-2-ylamino)-2-cyclohexylmethyl-butyricacid of the invention.

FIG. 2 shows a differential scanning calorimetry (DSC) trace (right sidevertical axis) and a thermal gravimetric analysis (TGA) trace (left sidevertical axis) of crystalline Form I(S)-4-((2S,3S)-7-carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydronaphthalen-2-ylamino)-2-cyclohexylmethyl-butyricacid of the invention.

FIG. 3 shows a dynamic moisture sorption (DMS) trace of crystalline FormI(S)-4-((2S,3S)-7-carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydronaphthalen-2-ylamino)-2-cyclohexylmethyl-butyricacid of the invention.

FIG. 4 shows an x-ray powder diffraction (XRPD) pattern of crystallineForm II(S)-4-((2S,3S)-7-carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydronaphthalen-2-ylamino)-2-cyclohexylmethyl-butyricacid of the invention.

FIG. 5 shows a differential scanning calorimetry (DSC) trace ofcrystalline Form II(S)-4-((2S,3S)-7-carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydronaphthalen-2-ylamino)-2-cyclohexylmethyl-butyricacid of the invention.

FIG. 6 shows an x-ray powder diffraction (XRPD) pattern of a crystallinehydrochloride salt of(S)-4-((2S,3S)-7-carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydronaphthalen-2-ylamino)-2-cyclohexylmethyl-butyricacid of the invention.

FIG. 7 shows a differential scanning calorimetry (DSC) trace (right sidevertical axis) and a thermal gravimetric analysis (TGA) trace (left sidevertical axis) of a crystalline hydrochloride salt of(S)-4-((2S,3S)-7-carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydronaphthalen-2-ylamino)-2-cyclohexylmethyl-butyricacid of the invention.

FIG. 8 shows an x-ray powder diffraction (XRPD) pattern of a crystallinehydrochloride salt of(S)-4-((2S,3S)-7-carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydronaphthalen-2-ylamino)-2-cyclohexylmethyl-butyricacid benzyl ester of the invention.

FIG. 9 shows a differential scanning calorimetry (DSC) trace of acrystalline hydrochloride salt of(S)-4-((2S,3S)-7-carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydronaphthalen-2-ylamino)-2-cyclohexylmethyl-butyricacid benzyl ester of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides crystalline solid forms of(S)-4-((2S,3S)-7-carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydronaphthalen-2-ylamino)-2-cyclohexylmethyl-butyricacid (1)

DEFINITIONS

When describing the compounds, compositions and methods of theinvention, the following terms have the following meanings, unlessotherwise indicated.

The term “therapeutically effective amount” means an amount sufficientto effect treatment when administered to a patient in need of treatment.

The term “treatment” as used herein means the treatment of a disease,disorder, or medical condition in a patient, such as a mammal(particularly a human) which includes, one or more of the followingactivities:

-   -   (a) preventing the disease, disorder, or medical condition from        occurring, i.e., prophylactic treatment of a patient;    -   (b) ameliorating the disease, disorder, or medical condition,        i.e., eliminating or causing regression of the disease,        disorder, or medical condition in a patient, including        counteracting the effects of other therapeutic agents;    -   (c) suppressing the disease, disorder, or medical condition,        i.e., slowing or arresting the development of the disease,        disorder, or medical condition in a patient; or    -   (d) alleviating the symptoms of the disease, disorder, or        medical condition in a patient.

It must be noted that, as used in the specification and appended claims,the singular forms “a”, “an”, “one”, and “the” may include pluralreferences, unless the content clearly dictates otherwise.

Naming Convention

Compound 1, is designated(S)-4-((2S,3S)-7-carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydronaphthalen-2-ylamino)-2-cyclohexylmethyl-butyricacid according to the IUPAC conventions as implemented in AutoNomsoftware, (MDL Information Systems, GmbH, Frankfurt, Germany). Thebicyclic 1,2,3,4-tetrahydronaphthalen-2-ylamino group is alternativelyknown by the common name, “aminotetralin”.

Crystalline Forms of the Invention

In one aspect, the invention provides crystalline(S)-4-((2S,3S)-7-carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydronaphthalen-2-ylamino)-2-cyclohexylmethyl-butyricacid (1) in two distinct forms.

In one aspect, Form I crystalline compound 1 is characterized by anx-ray powder diffraction (XRPD) pattern having diffraction peaks at 2θvalues of 6.92±0.20 and 15.34±0.20, and two or more diffraction peaks,including three or more and four or more diffraction peaks, at 2θ valuesselected from 10.24±0.20, 11.48±0.20, 12.32±0.20, 13.46±0.20,14.04±0.20, 17.30±0.20, 18.06±0.20, 20.30±0.20, 21.42±0.20, 23.48±0.20,25.54±0.20, 26.96±0.20, 29.30±0.20, and 30.72±0.20. In particular, inthis aspect, Form I is characterized by an x-ray powder diffractionpattern having three or more diffraction peaks, including four or morediffraction peaks, at 2θ values selected from 6.92±0.20, 10.24±0.20,13.46±0.20, 15.34±0.20, 18.06±0.20, and 21.42±0.20. Crystalline Form Iis further characterized by an x-ray powder diffraction pattern havingdiffraction peaks, at 2θ values of 6.92±0.20, 10.24±0.20, 13.46±0.20,15.34±0.20, 18.06±0.20, and 21.42±0.20.

As is well known in the field of powder x-ray diffraction, peakpositions of XRPD spectra are relatively less sensitive to experimentaldetails, such as details of sample preparation and instrument geometry,than are the relative peak heights. Thus, in one aspect, Form I ofcrystalline compound 1 is characterized by an x-ray powder diffractionpattern in which the peak positions are substantially in accordance withthose shown in FIG. 1.

The structure of crystalline Form I has been further characterized bysingle crystal x-ray diffraction analysis, providing the followinglattice parameters: unit cell is orthorhombic with dimensions a=7.546 Å,b=17.003 Å, c=20.628 Å, cell volume (V) of 2646.7 Å³; calculated densityis 1.151 g/cm³; space group is P2₁2₁2₁. The resulting molecularstructure confirms the asymmetric unit cell does not contain water orother solvent molecules and is consistent with the stereochemistry asdepicted above. The C—O bond distances of the carboxylic group as wellas the bond lengths and bond angles around the amine nitrogen areconsistent with compound 1 in crystalline Form I being a zwitterionicmolecule in which a proton has been transferred from the carboxylic acidto the amine nitrogen as represented schematically below:

X-ray powder diffraction peaks predicted from the derived atomicpositions are in excellent agreement with the observed XRPD pattern.

In another aspect, crystalline Form I is characterized by its behaviorwhen exposed to high temperature. As demonstrated in FIG. 2, thedifferential scanning calorimetry (DSC) trace exhibits a peak inendothermic heat flow, identified as a melt transition, in the range ofabout 162° C. to about 170° C., including between about 164° C. andabout 168° C. The thermal gravimetric analysis (TGA) trace shows nosignificant weight loss at temperatures below the melting point and astep profile consistent with a loss of one mole of water per mole ofcompound 1 at a temperature around the melting point. The release ofwater can be attributed to a chemical degradation reaction.

Crystalline Form I has been demonstrated to have a reversiblesorption/desorption profile with a small propensity for hygroscopicity.Form I demonstrated less than about 2.5% weight gain in the humidityrange of 2% to 90% relative humidity at room temperature, as shown inFIG. 3. In particular, Form I demonstrated less than about 1% weightgain in the range of 40% to 75% relative humidity, the humidity range atwhich oral formulations are typically manufactured.

In another aspect, the invention provides compound 1 in crystalline FormII. Crystalline Form II is identified by the XRPD pattern of FIG. 4 andthe DSC profile of FIG. 5. In one aspect, crystalline Form II ischaracterized by an x-ray powder diffraction (XRPD) pattern havingdiffraction peaks at 2θ values of 9.05±0.20 and 16.52±0.20, and havingtwo or more diffraction peaks, including three or more and four or morediffraction peaks, at 2θ values selected from 9.80±0.20, 12.44±0.20,12.92±0.20, 14.21±0.20, 15.62±0.20, 17.27±0.20, 19.04±0.20, 19.85±0.20,21.29±0.20, 22.43±0.20, 23.48±0.20, 23.99±0.20, and 26.09±0.20. Inparticular, in this aspect, Form II is characterized by an x-ray powderdiffraction pattern having two or more diffraction peaks, includingthree or more and four or more diffraction peaks, at 2θ values selectedfrom 9.05±0.20, 9.80±0.20, 12.44±0.20, 12.92±0.20, 16.52±0.20,23.99±0.20, and 26.09±0.20. Form II is still further characterized by anx-ray powder diffraction pattern having diffraction peaks at 9.05±0.20,9.80±0.20, 12.44±0.20, 12.92±0.20, 16.52±0.20, 23.99±0.20, and26.09±0.20. In yet another aspect, crystalline Form II is characterizedby an x-ray powder diffraction pattern in which the peak positions aresubstantially in accordance with those shown in FIG. 4.

The thermal behavior exhibited in FIG. 5 is consistent with theidentification of crystalline Form II as a metastable form whichundergoes a transformation at a temperature around 114° C. to 115° C.and has a melting point accompanied by chemical degradation at atemperature around 157° C.

In still another aspect, the invention provides a crystallinehydrochloride salt of compound 1.

In one aspect, a crystalline hydrochloride salt of the present inventionis characterized by an x-ray powder diffraction (XRPD) pattern havingtwo or more diffraction peaks, including three or more and four or morediffraction peaks, at 2θ values selected from 6.80±0.20, 9.80±0.20,12.71±0.20, 13.31±0.20, 15.14±0.20, 19.97±0.20, 21.44±0.20, 22.64±0.20,23.27±0.20, 24.44±0.20, and 25.37±0.20. In another aspect, a crystallinehydrochloride salt of compound 1 is characterized by an x-ray powderdiffraction pattern in which the peak positions are substantially inaccordance with those shown in FIG. 6.

The crystalline hydrochloride salt is also characterized by itsdifferential scanning calorimetry (DSC) trace which exhibits a firstpeak in endothermic heat flow in the range of about 185° C. to about193° C., identified as a melting transition, and a second peak in therange of about 220° C. to about 140° C. that is understood to conform toa degradation event, as illustrated in FIG. 7. Samples of thecrystalline hydrochloride salt prepared in Examples 8, 9, and 10retained their appearance and flowability when exposed to relativehumidity in the range of about 2% relative humidity to about 90%relative humidity at room temperature.

These properties of the crystalline forms of this invention are furtherillustrated in the Examples below.

Synthetic Procedures and Intermediates

Compound 1,(S)-4-((2S,3S)-7-carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydronaphthalen-2-ylamino)-2-cyclohexylmethyl-butyricacid can be prepared from readily available starting materials in solidamorphous form using the procedures described in the Examples below, orusing the procedures described in the commonly-assigned U.S.applications listed in the Background section of this application.

In one method of preparation, crystalline Form I of the invention isprepared by dissolving amorphous compound 1 in a polar diluent to form acrystallization process mixture, and holding the process mixture forbetween about 12 hours and about 4 days. Typically the crystallizationprocess is conducted at about ambient temperature. Suitable diluentsinclude methanol, isopropanol, 1-propanol, and acetonitrile, andmixtures of one or more of the foregoing with water. Exemplary diluentsystems include a mixture of acetonitrile, methanol and water, forexample, about 69% acetonitrile, about 21% methanol and about 10% water;a mixture of 1-propanol, acetonitrile, and water, in particular about95% to about 97% (2:1 1-propanol:acetonitrile) and about 5% to about 3%water; a mixture of methanol and water, for example, about 90% methanoland about 10% water; and a mixture of acetonitrile and water, forexample about 84% acetonitrile and about 16% water. Compound 1 istypically present in the process mixture at a concentration of betweenabout 150 mg/mL and about 700 mg/mL.

When water is not present in the diluent system, it is useful to holdthe process mixture for a time period at the upper end of the statedrange (See, Example 4 below, where crystallization was allowed toproceed over 4 days.) A useful process, therefore, for preparingcrystalline Form I comprises dissolving compound 1 in a diluentcomprising a polar diluent and between about 3% and about 20% water toform a process mixture; holding the process mixture for at least about12 hours; and recovering the resulting crystals.

Upon completion of the reaction, crystalline Form I is isolated from theprocess mixture by any conventional means, such as filtration,concentration, centrifugation, and the like.

In another method of preparation, crystalline Form I is advantageouslyprepared directly from a protected precursor 2 of compound 1, accordingto the following process:

As summarized in Scheme A, benzyl-protected aldehyde 4 is reacted withaminotetralin intermediate 3 to provide a benzyl-protected precursor 2,which is deprotected to provide compound 1 in crystalline Form I. Thealdehyde reagent 4 is conveniently prepared in situ from thecorresponding bisulfite adduct 5.

In a typical process, a solution of between about 1 and about 1.5equivalents of aldehyde 4 in an inert diluent is generated by treatingthe bisulfite adduct 5 with an equal number of equivalents of sodiumhydroxide. The aldehyde 4 is then contacted with aminotetralin 3 andbetween about 1 and about 3 equivalents of a reducing agent, such assodium triacetoxyborohydride. Aminotetralin 3 may be provided as an acidsalt, typically a hydrochloride salt. The reaction is typicallyconducted at a temperature between about 0° C. and about 30° C. forbetween about 2 and about 24 hours or until the reaction issubstantially complete. The protected intermediate 2 is convenientlyisolated in solid form as a crystalline hydrochloride salt.

Finally, intermediate 2 is debenzylated by catalytic hydrogenolysis,typically using a transition metal catalyst, for example, palladium orplatinum, to provide crystalline Form I. When intermediate 2 is providedin salt form, the neutral form of the intermediate is first generated insitu by treatment with base. The reaction is conducted in a diluentchosen to be both compatible with hydrogenolysis and conducive tocrystallization. Mixtures comprising water and another polar diluent,such as methanol, isopropanol, 1-propanol, acetonitrile, and/ordimethylformamide, are suitable for this reaction. Mixtures comprisingbetween about 10% and about 20% water, for example, a mixture ofacetonitrile and between about 10% and about 20% water, areadvantageously used in the debenzylation reaction. The reaction product,crystalline Form I, can be isolated by conventional means, such asfiltration.

Accordingly, in a method aspect, among other processes, the inventionprovides a process for preparing crystalline Form I of compound 1, theprocess comprising: deprotecting the benzyl-protected intermediate 2 bycatalytic hydrogenolysis in the presence of a polar diluent comprisingbetween about 10% and about 20% water to form crystalline Form I.

In an additional method aspect, the invention provides a process forpreparing crystalline Form I of compound 1, the process comprising (a)reacting protected aldehyde 4 with aminotetralin 3 to provide protectedintermediate 2, and (b) deprotecting the benzyl-protected intermediate 2by catalytic hydrogenolysis in the presence of a polar diluentcomprising between about 10% and about 20% water to form crystallineForm I.

Deprotection of intermediate 2 in a diluent that is compatible withhydrogenolysis but that does not promote crystallization, such as, ethylacetate, tetrahydrofuran, or methyl-tetrahydrofuran, results in thepreparation of compound 1, albeit not necessarily in crystalline form.In yet another method aspect, the invention provides a process forpreparing compound 1, the process comprising (a) reacting protectedaldehyde 4 with aminotetralin 3 to provide protected intermediate 2, and(b) deprotecting the benzyl-protected intermediate 2 by catalytichydrogenolysis to provide compound 1.

Further, in a composition aspect, the invention provides thebenzyl-protected aldehyde 4, (S)-2-cyclohexylmethyl-4-oxo-butyric acidbenzyl ester, and the bisulfite adduct 5, sodium(S)-3-benzyloxycarbonyl-4-cyclohexyl-1-hydroxy-butane-1-sulfonate usefulfor preparing compound 1. As described in Preparation 8, thebenzyl-protected bisulfite adduct 5 can be prepared from thecorresponding methyl ester bisulfite, sodium(S)-4-cyclohexyl-1-hydroxy-3-methoxycarbonyl-butane-1-sulfonate,synthesis of which is described in Preparation 7. Alternatively,bisulfite adduct 5 could be prepared by processes analogous to those ofPreparation 7 through analogous benzyl-protected carboxylic acid (5a)and alcohol (5b) intermediates:

In yet another composition aspect, the invention provides thebenzyl-protected precursor 2,(S)-4-((2S,3S)-7-carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydronaphthalen-2-ylamino)-2-cyclohexylmethyl-butyricacid benzyl ester and the hydrochloride salt, thereof. The crystallinehydrochloride salt of compound 2 is characterized by the XRPD pattern ofFIG. 8 and the DSC profile of FIG. 9 which exhibits a peak inendothermic heat flow, consistent with a melting point, at a temperaturebetween about 205° C. and about 210° C. Pharmaceutical intermediates incrystalline form are desirable both due to the typical purification thatresults from crystallization and due to the expected greater stabilityon storage as compared with non-crystalline material.

As described in the Examples below, crystalline Form II is prepared bydissolving amorphous compound 1 in a polar diluent comprising alcohol,for example, isopropanol, or isopropanol and acetonitrile at aconcentration between about 150 mg/mL and about 700 mg/mL to form acrystallization process mixture, and holding the process mixture forless than about a day. Optionally an anti-solvent, for example 1:1acetonitrile:ethyl acetate can be added to the process mixture beforethe holding period to promote crystallization from solution.

The crystalline hydrochloride salt of the present invention isadvantageously prepared from the amorphous hydrochloride salt ofcompound 1. The amorphous salt of compound 1 is dispersed in amoderately polar solvent such as ethyl acetate or diethylene glycoldimethyl ether, at a concentration between about 20 mg/mL and about 350mg/mL, optionally with heating followed by slow cooling, and then heldfor a period of between about 3 days and about 12 days. The resultingcrystals can be isolated conventionally.

Pharmaceutical Compositions

The crystalline solid forms of the invention are typically administeredto a patient in the form of a pharmaceutical composition or formulation.Such pharmaceutical compositions may be administered to the patient byany acceptable route of administration including, but not limited to,oral, rectal, vaginal, nasal, inhaled, topical (including transdermal)and parenteral modes of administration.

Accordingly, in one of its compositions aspects, the invention isdirected to a pharmaceutical composition comprising apharmaceutically-acceptable carrier or excipient and a therapeuticallyeffective amount of a crystalline solid form of compound 1 or acrystalline hydrochloride salt of compound 1. Optionally, suchpharmaceutical compositions may contain other therapeutic and/orformulating agents if desired. When discussing compositions, it isunderstood the term “solid form of the invention” includes thecrystalline Forms I and II of compound 1 as well as a crystallinehydrochloride salt of compound 1.

The pharmaceutical compositions of the invention typically contain atherapeutically effective amount of the active agent. Those skilled inthe art will recognize, however, that a pharmaceutical composition maycontain more than a therapeutically effective amount, i.e., bulkcompositions, or less than a therapeutically effective amount, i.e.,individual unit doses designed for multiple administration to achieve atherapeutically effective amount.

Typically, such pharmaceutical compositions will contain from about 0.1to about 95% by weight of the active agent; preferably, from about 5 toabout 70% by weight; and more preferably from about 10 to about 60% byweight of the active agent.

Any conventional carrier or excipient may be used in the pharmaceuticalcompositions of the invention. The choice of a particular carrier orexcipient, or combinations of carriers or excipients, will depend on themode of administration being used to treat a particular patient or typeof medical condition or disease state. In this regard, the preparationof a suitable pharmaceutical composition for a particular mode ofadministration is well within the scope of those skilled in thepharmaceutical arts. Additionally, the carriers or excipients used inthe pharmaceutical compositions of this invention arecommercially-available. By way of further illustration, conventionalformulation techniques are described in Remington: The Science andPractice of Pharmacy, 20^(th) Edition, Lippincott Williams & White,Baltimore, Md. (2000); and H. C. Ansel et al., Pharmaceutical DosageForms and Drug Delivery Systems, 7^(th) Edition, Lippincott Williams &White, Baltimore, Md. (1999).

Representative examples of materials which can serve as pharmaceuticallyacceptable carriers include, but are not limited to, the following:sugars, such as lactose, glucose and sucrose; starches, such as cornstarch and potato starch; cellulose, such as microcrystalline cellulose,and its derivatives, such as sodium carboxymethyl cellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin;talc; excipients, such as cocoa butter and suppository waxes; oils, suchas peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil,corn oil and soybean oil; glycols, such as propylene glycol; polyols,such as glycerin, sorbitol, mannitol and polyethylene glycol; esters,such as ethyl oleate and ethyl laurate; agar; buffering agents, such asmagnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-freewater; isotonic saline; Ringer's solution; ethyl alcohol; phosphatebuffer solutions; and other non-toxic compatible substances employed inpharmaceutical compositions.

Pharmaceutical compositions are typically prepared by thoroughly andintimately mixing or blending the active agent with apharmaceutically-acceptable carrier and one or more optionalingredients. The resulting uniformly blended mixture can then be shapedor loaded into tablets, capsules, pills and the like using conventionalprocedures and equipment.

The pharmaceutical compositions of the invention are preferably packagedin a unit dosage form. The term “unit dosage form” refers to aphysically discrete unit suitable for dosing a patient, i.e., each unitcontaining a predetermined quantity of active agent calculated toproduce the desired therapeutic effect either alone or in combinationwith one or more additional units. For example, such unit dosage formsmay be capsules, tablets, pills, and the like, or unit packages suitablefor parenteral administration.

In one embodiment, the pharmaceutical compositions of the invention aresuitable for oral administration. Suitable pharmaceutical compositionsfor oral administration may be in the form of capsules, tablets, pills,lozenges, cachets, dragees, powders, granules; or as a solution or asuspension in an aqueous or non-aqueous liquid; or as an oil-in-water orwater-in-oil liquid emulsion; or as an elixir or syrup; and the like;each containing a predetermined amount of a compound of the presentinvention as an active ingredient.

When intended for oral administration in a solid dosage form (i.e., ascapsules, tablets, pills and the like), the pharmaceutical compositionsof the invention will typically comprise the active agent and one ormore pharmaceutically-acceptable carriers, such as sodium citrate ordicalcium phosphate. Optionally or alternatively, such solid dosageforms may also comprise: fillers or extenders, such as starches,microcrystalline cellulose, lactose, sucrose, glucose, mannitol, and/orsilicic acid; binders, such as carboxymethylcellulose, alginates,gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; humectants, suchas glycerol; disintegrating agents, such as agar-agar, calciumcarbonate, potato or tapioca starch, alginic acid, certain silicates,and/or sodium carbonate; solution retarding agents, such as paraffin;absorption accelerators, such as quaternary ammonium compounds; wettingagents, such as cetyl alcohol and/or glycerol monostearate; absorbents,such as kaolin and/or bentonite clay; lubricants, such as talc, calciumstearate, magnesium stearate, solid polyethylene glycols, sodium laurylsulfate, and/or mixtures thereof; coloring agents; and buffering agents.

Release agents, wetting agents, coating agents, sweetening, flavoringand perfuming agents, preservatives and antioxidants can also be presentin the pharmaceutical compositions of the invention. Examples ofpharmaceutically-acceptable antioxidants include: water-solubleantioxidants, such as ascorbic acid, cysteine hydrochloride, sodiumbisulfate, sodium metabisulfate, sodium sulfite and the like;oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole, butylated hydroxytoluene, lecithin, propyl gallate,alpha-tocopherol, and the like; and metal-chelating agents, such ascitric acid, ethylenediamine tetraacetic acid, sorbitol, tartaric acid,phosphoric acid, and the like. Coating agents for tablets, capsules,pills and like, include those used for enteric coatings, such ascellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropylmethylcellulose phthalate, methacrylic acid-methacrylic acid estercopolymers, cellulose acetate trimellitate, carboxymethyl ethylcellulose, hydroxypropyl methyl cellulose acetate succinate, and thelike.

Pharmaceutical compositions of the invention may also be formulated toprovide slow or controlled release of the active agent using, by way ofexample, hydroxypropyl methyl cellulose in varying proportions; or otherpolymer matrices, liposomes and/or microspheres. In addition, thepharmaceutical compositions of the invention may optionally containopacifying agents and may be formulated so that they release the activeingredient only, or preferentially, in a certain portion of thegastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active agent can also be in micro-encapsulated form, ifappropriate, with one or more of the above-described excipients.

Suitable liquid dosage forms for oral administration include, by way ofillustration, pharmaceutically-acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. Liquid dosage formstypically comprise the active agent and an inert diluent, such as, forexample, water or other solvents, solubilizing agents and emulsifiers,such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, oils (esp., cottonseed, groundnut, corn, germ, olive, castor andsesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof. Suspensions, inaddition to the active ingredient, may contain suspending agents suchas, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

The solid forms of this invention can also be administered parenterally(e.g. by intravenous, subcutaneous, intramuscular or intraperitonealinjection). For parenteral administration, the active agent is typicallyadmixed with a suitable vehicle for parenteral administration including,by way of example, sterile aqueous solutions, saline, low molecularweight alcohols such as propylene glycol, polyethylene glycol, vegetableoils, gelatin, fatty acid esters such as ethyl oleate, and the like.Parenteral formulations may also contain one or more anti-oxidants,solubilizers, stabilizers, preservatives, wetting agents, emulsifiers,or dispersing agents. These formulations may be rendered sterile by useof a sterile injectable medium, a sterilizing agent, filtration,irradiation, or heat.

Alternatively, the pharmaceutical compositions of the invention areformulated for administration by inhalation. Suitable pharmaceuticalcompositions for administration by inhalation will typically be in theform of an aerosol or a powder. Such compositions are generallyadministered using well-known delivery devices, such as a metered-doseinhaler, a dry powder inhaler, a nebulizer or a similar delivery device.

When administered by inhalation using a pressurized container, thepharmaceutical compositions of the invention will typically comprise theactive ingredient and a suitable propellant, such asdichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas.Additionally, the pharmaceutical composition may be in the form of acapsule or cartridge (made, for example, from gelatin) comprising acompound of the invention and a powder suitable for use in a powderinhaler. Suitable powder bases include, by way of example, lactose orstarch.

The solid forms of the invention can also be administered transdermallyusing known transdermal delivery systems and excipients. For example,the active agent can be admixed with permeation enhancers, such aspropylene glycol, polyethylene glycol monolaurate, azacycloalkan-2-onesand the like, and incorporated into a patch or similar delivery system.Additional excipients including gelling agents, emulsifiers and buffers,may be used in such transdermal compositions if desired.

If desired, the solid forms of this invention may be administered incombination with one or more other therapeutic agents. In thisembodiment, a solid form of this invention is either physically mixedwith the other therapeutic agent to form a composition containing bothagents; or each agent is present in separate and distinct compositionswhich are administered to the patient simultaneously or sequentially.

For example, a solid form of the invention can be combined with secondtherapeutic agent using conventional procedures and equipment to form acomposition comprising a compound 1 and a second therapeutic agent.Additionally, the therapeutic agents may be combined with apharmaceutically acceptable carrier to form a pharmaceutical compositioncomprising a salt of the invention, a second therapeutic agent and apharmaceutically acceptable carrier. In this embodiment, the componentsof the composition are typically mixed or blended to create a physicalmixture. The physical mixture is then administered in a therapeuticallyeffective amount using any of the routes described herein.Alternatively, the therapeutic agents may remain separate and distinctbefore administration to the patient. In this embodiment, the agents arenot physically mixed together before administration but are administeredsimultaneously or at separate times as separate compositions. Suchcompositions can be packaged separately or may be packaged together as akit. The two therapeutic agents in the kit may be administered by thesame route of administration or by different routes of administration.

Any therapeutic agent compatible with the present active agent may beused as the second therapeutic agent. In particular, prokinetic agentsacting via mechanisms other than mu opioid receptor antagonism may beused in combination with the present compounds. For example, 5-HT₄receptor agonists, such as tegaserod, renzapride, mosapride,prucalopride, 1-isopropyl-1H-indazole-3-carboxylic acid{(1S,3R,5R)-8-[2-(4-acetylpiperazin-1-yl)ethyl]-8-azabicyclo[3.2.1]oct-3-yl}amide,1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[(R)-2-hydroxy-3-(methanesulfonyl-methyl-amino)propyl]-8-azabicyclo[3.2.1]oct-3-yl}amide,or4-(4-{[(2-isopropyl-1H-benzoimidazole-4-carbonyl)amino]methyl}-piperidin-1-ylmethyl)piperidine-1-carboxylicacid methyl ester may be used as the second therapeutic agent.

Additional useful prokinetic agents include, but are not limited to,5-HT₃ receptor agonists (e.g. pumosetrag), 5-HT_(1A) receptorantagonists (e.g. AGI 001), alpha-2-delta ligands (e.g. PD-217014),chloride channel openers (e.g. lubiprostone), dopamine antagonists (e.g.itopride, metaclopramide, domperidone), GABA-B agonists (e.g. baclofen,AGI 006), kappa opioid agonists (e.g. asimadoline), muscarinic M₁ and M₂antagonists (e.g. acotiamide), motilin agonists (e.g. mitemcinal),guanylate cyclase activators (e.g. MD-1100) and ghrelin agonists (e.g.Tzp 101, RC 1139).

In addition, the solid forms of the invention can be combined withopioid therapeutic agents. Such opioid agents include, but are notlimited to, morphine, pethidine, codeine, dihydrocodeine, oxycontin,oxycodone, hydrocodone, sufentanil, fentanyl, remifentanil,buprenorphine, methadone, and heroin.

Numerous additional examples of such therapeutic agents are known in theart and any such known therapeutic agents may be employed in combinationwith the compounds of this invention. Secondary agent(s), when included,are present in a therapeutically effective amount, i.e. in any amountthat produces a therapeutically beneficial effect when co-administeredwith a compound of the invention. Suitable doses for the othertherapeutic agents administered in combination with a compound of theinvention are typically in the range of about 0.05 μg/day to about 100mg/day.

Accordingly, the pharmaceutical compositions of the invention optionallyinclude a second therapeutic agent as described above.

The following examples illustrate representative pharmaceuticalcompositions of the present invention:

Formulation Example A Hard Gelatin Capsules for Oral Administration

A solid form of the invention (50 g), spray-dried lactose (200 g) andmagnesium stearate (10 g) are thoroughly blended. The resultingcomposition is loaded into a hard gelatin capsule (260 mg of compositionper capsule).

Formulation Example B Hard Gelatin Capsules for Oral Administration

A solid form of the invention (20 mg), starch (89 mg), microcrystallinecellulose (89 mg), and magnesium stearate (2 mg) are thoroughly blendedand then passed through a No. 45 mesh U.S. sieve. The resultingcomposition is loaded into a hard gelatin capsule (200 mg of compositionper capsule).

Formulation Example C Gelatin Capsules for Oral Administration

A solid form of the invention (10 mg), polyoxyethylene sorbitanmonooleate (50 mg), and starch powder (250 mg) are thoroughly blendedand then loaded into a gelatin capsule (310 mg of composition percapsule).

Formulation Example D Tablets for Oral Administration

A solid form of the invention (5 mg), starch (50 mg), andmicrocrystalline cellulose (35 mg) are passed through a No. 45 mesh U.S.sieve and mixed thoroughly. A solution of polyvinylpyrrolidone (10 wt %in water, 4 mg) is mixed with the resulting powders, and this mixture isthen passed through a No. 14 mesh U.S. sieve. The granules so producedare dried at 50-60° C. and passed through a No. 18 mesh U.S. sieve.Sodium carboxymethyl starch (4.5 mg), magnesium stearate (0.5 mg) andtalc (1 mg), which have previously been passed through a No. 60 meshU.S. sieve, are then added to the granules. After mixing, the mixture iscompressed on a tablet machine to afford a tablet weighing 100 mg.

Formulation Example E Tablets for Oral Administration

A solid form of the invention (25 mg), microcrystalline cellulose (400mg), fumed silicon dioxide (10 mg), and stearic acid (5 mg) arethoroughly blended and then compressed to form tablets (440 mg ofcomposition per tablet).

Formulation Example F Single-Scored Tablets for Oral Administration

A solid form of the invention (15 mg), cornstarch (50 mg),croscarmellose sodium (25 mg), lactose (120 mg), and magnesium stearate(5 mg) are thoroughly blended and then compressed to form single-scoredtablet (215 mg of compositions per tablet).

Formulation Example G Suspension for Oral Administration

The following ingredients are thoroughly mixed to form a suspension fororal administration containing 100 mg of active ingredient per 10 mL ofsuspension:

Ingredients Amount Solid form of the invention 0.1 g Fumaric acid 0.5 gSodium chloride 2.0 g Methyl paraben 0.15 g Propyl paraben 0.05 gGranulated sugar 25.5 g Sorbitol (70% solution) 12.85 g Veegum k(Vanderbilt Co.) 1.0 g Flavoring 0.035 mL Colorings 0.5 mg Distilledwater q.s. to 100 mL

Formulation Example H Dry Powder Composition

A micronized solid form of the invention (1 mg) is blended with lactose(25 mg) and then loaded into a gelatin inhalation cartridge. Thecontents of the cartridge are administered using a powder inhaler.

Formulation Example J Injectable Formulation

A solid form of the invention (0.1 g) is blended with 0.1 M sodiumcitrate buffer solution (15 mL). The pH of the resulting solution isadjusted to pH 6 using 1 N aqueous hydrochloric acid or 1 N aqueoussodium hydroxide. Sterile normal saline in citrate buffer is then addedto provide a total volume of 20 mL.

It will be understood that any solid form of the invention, (i.e.crystalline Form I or Form II, or crystalline hydrochloride salt) thatis suitable for the particular mode of administration, can be used inthe pharmaceutical compositions discussed above.

Utility

The present active agent,(S)-4-((2S,3S)-7-carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydronaphthalen-2-ylamino)-2-cyclohexylmethyl-butyricacid, is an antagonist at the mu opioid receptor and therefore thecrystalline solid forms of the invention are expected to be useful fortreating medical conditions mediated by mu opioid receptors orassociated with mu opioid receptor activity, i.e. medical conditionswhich are ameliorated by treatment with a mu opioid receptor antagonist.In particular, the solid forms of the invention are expected to beuseful for treating adverse effects associated with use of opioidanalgesics, i.e. symptoms such as constipation, decreased gastricemptying, abdominal pain, bloating, nausea, and gastroesophageal reflux,termed collectively opioid-induced bowel dysfunction. The present solidforms are also expected to be useful for treating post-operative ileus,a disorder of reduced motility of the gastrointestinal tract that occursafter abdominal or other surgery. In addition, it has been suggestedthat mu opioid receptor antagonist compounds, such as compound 1 may beused for reversing opioid-induced nausea and vomiting.

Since compound 1 has been shown to increase motility of thegastrointestinal (GI) tract in animal models, the solid forms of theinvention are expected to be useful for treating disorders of the GItract caused by reduced motility in mammals, including humans. Such GImotility disorders include, by way of illustration, chronicconstipation, constipation-predominant irritable bowel syndrome (C-IBS),diabetic and idiopathic gastroparesis, and functional dyspepsia.

In one aspect, therefore, the invention provides a method of increasingmotility of the gastrointestinal tract in a mammal, the methodcomprising administering to the mammal a therapeutically effectiveamount of a pharmaceutical composition comprising apharmaceutically-acceptable carrier and a solid form of the invention.

When used to treat disorders of reduced motility of the GI tract orother conditions mediated by mu opioid receptors, the solid forms of theinvention will typically be administered orally in a single daily doseor in multiple doses per day, although other forms of administration maybe used. For example, particularly when used to treat post-operativeileus, the solid forms of the invention may be administeredparenterally. The amount of active agent administered per dose or thetotal amount administered per day will typically be determined by aphysician, in the light of the relevant circumstances, including thecondition to be treated, the chosen route of administration, the actualcompound administered and its relative activity, the age, weight, andresponse of the individual patient, the severity of the patient'ssymptoms, and the like.

Suitable doses for treating disorders of reduced motility of the GItract or other disorders mediated by mu opioid receptors will range fromabout 0.0007 to about 20 mg/kg/day of active agent, including from about0.0007 to about 1.4 mg/kg/day. For an average 70 kg human, this wouldamount to from about 0.05 to about 100 mg per day of active agent.

In one aspect of the invention, the solid forms of the invention areused to treat opioid-induced bowel dysfunction. When used to treatopioid-induced bowel dysfunction, the solid forms of the invention willtypically be administered orally in a single daily dose or in multipledoses per day. Preferably, the dose for treating opioid-induced boweldysfunction will range from about 0.05 to about 100 mg per day.

In another aspect of the invention, the solid forms of the invention areused to treat post-operative ileus. When used to treat post-operativeileus, the solid forms of the invention will typically be administeredorally or intravenously in a single daily dose or in multiple doses perday. Preferably, the dose for treating post-operative ileus will rangefrom about 0.05 to about 100 mg per day.

The invention also provides a method of treating a mammal having adisease or condition associated with mu opioid receptor activity, themethod comprising administering to the mammal a therapeuticallyeffective amount of a solid form of the invention or of a pharmaceuticalcomposition comprising a solid form of the invention.

The present active agent is optionally administered in combination withanother therapeutic agent or agents, in particular, in combination withprokinetic agents acting via non-mu opioid mechanisms. Accordingly, inanother aspect, the methods and compositions of the invention furthercomprise a therapeutically effective amount of another prokinetic agent.

As described above, solid forms of the invention are mu opioid receptorantagonists. The invention further provides, therefore, a method ofantagonizing a mu opioid receptor in a mammal, the method comprisingadministering a solid form of the invention to the mammal.

Among other properties, the present active agent has been found toexhibit potent binding to mu opioid receptors and little or no agonismin mu receptor functional assays. Therefore, the solid forms of theinvention are potent mu opioid receptor antagonists. Further, the activeagent has demonstrated predominantly peripheral activity as comparedwith central nervous system activity in animal models. Therefore, thesolid forms of the invention can be expected to reverse opioid-inducedreductions in GI motility without interfering with the beneficialcentral effects of analgesia. These properties, as well as the utilityof the compounds of the invention, can be demonstrated using various invitro and in vivo assays well-known to those skilled in the art.Representative assays are described in further detail in the followingexamples.

EXAMPLES

The following synthetic and biological examples are offered toillustrate the invention, and are not to be construed in any way aslimiting the scope of the invention. In the examples below, thefollowing abbreviations have the following meanings unless otherwiseindicated. Abbreviations not defined below have their generally acceptedmeanings

DCM dichloromethane

DMF=N,N-dimethylformamide

MeOH=methanol

MeTHF=2-methyltetrahydrofuran

MTBE=tert-butyl methyl ether

psi=pounds per square inch

RT=room temperature

Reagents and solvents were purchased from commercial suppliers (Aldrich,Fluka, Sigma, etc.), and used without further purification. Reactionswere run under nitrogen atmosphere, unless noted otherwise. Progress ofreaction mixtures was monitored by thin layer chromatography (TLC), highperformance liquid chromatography (HPLC), and mass spectrometry.Reaction mixtures were worked up as described specifically in eachreaction; commonly they were purified by extraction and otherpurification methods such as temperature-, and solvent-dependentprecipitation. Characterization of reaction products was routinelycarried out by mass and ¹H-NMR spectrometry. For NMR measurement,samples were dissolved in deuterated solvent (CD₃OD, CDCl₃, or DMSO-d₆),and ¹H-NMR spectra were acquired with a Varian Gemini 2000 instrument(400 MHz) under standard observation conditions. Mass spectrometricidentification of compounds was performed by an electrospray ionizationmethod (ESMS) with an Applied Biosystems (Foster City, Calif.) model API150 EX instrument or an Agilent (Palo Alto, Calif.) model 1200 LC/MSDinstrument. Water content was determined by Karl-Fischer titration usinga Brinkmann (Westbury, N.Y.) Metrohm Karl Fischer Model 813 coulometer.Purity was determined by HPLC using the following conditions:

Column: Zorbax SB-Aq, 5 μm. 4.6×250 mm

Column temperature: 40° C.

Flow rate: 1.0 mL/min

Mobile Phases: A=Water/ACN (98:2)+0.1% TFA

-   -   B=Water/ACN (10:90)+0.1% TFA,

Injection volume: 10 μL

Detector wavelength: 214 nm

Compounds were dissolved in Water/ACN (50:50) at about 1 mg/mL andanalyzed using the following gradient over 20 min (time (min)/% B):0/10, 2.5/20, 9/75, 15/90, 17/90, 18/10, 20/10.

Preparation 17,7-diethyl-5-hydroxy-1a,2,7,7a-tetrahydro-1-aza-cyclopropa[b]naphthalene-1-carboxylicacid tert-butyl ester a.7-Amino-6-bromo-8,8-diethyl-5,6,7,8-tetrahydronaphthalen-2-olhydrobromide

To a flask was added7,7-diethyl-5-methoxy-1a,2,7,7a-tetrahydro-1H-1-aza-cyclopropa[b]naphthalene(268 g, 1.16 mol) and hydrogen bromide (1.97 L, 17.38 mol), followed bytetra-N-butylammonium bromide (38 g, 0.12 mol). The reaction mixture washeated at 100° C. overnight with stirring, cooled to room temperatureand then poured into stirred ethyl acetate (2.5 L). The product wasisolated by filtration, the filter cake was washed with ethyl acetate(2×200 mL) and dried to yield crude product (370 g) as a purplish solid.The crude product was suspended in ethanol (1.50 L) then heated at 80°C. for 30 min. The resulting slurry was cooled to room temperature over1 h, and filtered. The flask and filter cake with were washed withethanol (2×100 mL) and then with ethyl acetate (100 mL) and driedovernight to yield the title compound (275 g, ˜96% purity).

b.7,7-Diethyl-5-hydroxy-1a,2,7,7a-tetrahydro-1-aza-cyclopropa[b]naphthalene-1-carboxylicacid tert-butyl ester

To a slurry of7-amino-6-bromo-8,8-diethyl-5,6,7,8-tetrahydronaphthalen-2-olhydrobromide (20.0 g, 52.8 mmol) and ethyl acetate (200 mL) was added1.0 M sodium hydroxide in water (106 mL). The reaction mixture wasstirred at 25° C. for 2 h, di-tert-butyldicarbonate (15 g, 68 mmol) inethyl acetate (5 mL) was added and the reaction mixture was stirred atroom temperature for 2 h. Following removal of two-thirds of the ethylacetate (135 mL), heptane (135 mL) was added and the resulting slurrywas stirred at room temperature over 30 min and then at 5° C. overnight.The slurry was filtered, and the filter cake was rinsed with water (100mL), rinsed with heptane (50 mL), and dried under vacuum to give thetitle compound (14.3 g).

Preparation 2trans-(7-Cyano-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydronaphthalen-2-yl)-carbamicacid tert-butyl ester* a.trans-(1,1-Diethyl-7-hydroxy-3-methoxy-1,2,3,4-tetrahydronaphthalen-2-yl)-carbamicacid tert-butyl ester

To a slurry of7,7-diethyl-5-hydroxy-1a,2,7,7a-tetrahydro-1-aza-cyclopropa[b]naphthalene-1-carboxylicacid tert-butyl ester (170.0 g, 535.6 mmol) and methanol (1700 mL) wasadded pyridinium p-toluenesulfonate (13.4 g, 53.6 mmol) and the reactionmixture was stirred at 40° C. for 4 h. The volume was reduced by rotaryevaporation to ˜300 mL resulting in a thick white slurry. The productwas isolated by filtration; the filter cake was washed with coldmethanol (50 mL) and dried in air for 3 h to yield the title compound(150 g). The filtrate was reduced to ˜50 mL and stirred at 0° C. for 2h, filtered, and dried to yield additional product (25 g). *In this andthe following examples, the prefix trans refers to a mixture of the(2S),(2S) diastereomer and the (2R),(2R) diastereomer.

b. trans-Trifluoro-methanesulfonic acid7-tert-butoxycarbonylamino-8,8-diethyl-6-methoxy-5,6,7,8-tetrahydronaphthalen-2-ylester

A mixture oftrans-(1,1-diethyl-7-hydroxy-3-methoxy-1,2,3,4-tetrahydronaphthalen-2-yl)-carbamicacid tert-butyl ester (195.0 g, 0.558 mol), triethylamine (160 mL, 1.1mol) and ethyl acetate (2000 mL) was stirred at room temperature for 15min and cooled to 0° C. followed by slow addition oftrifluoro-methanesulfonyl chloride (150 g, 0.89 mol) keeping theinternal temperature below 4° C. The resulting slurry was stirred at 0°C. for 1 h. Additional triethylamine (16 mL) followed by additionaltrifluoromethanesulfonyl chloride (15.0 g) was added slowly maintaininga temperature below 5° C. The reaction mixture was stirred at roomtemperature for an additional hour. Diluted brine (1.0 L) was added andthe reaction mixture was stirred for 10 min at room temperature. Thelayers were separated; the organic layer was washed with diluted NaHCO₃(1.0 L) and then concentrated to ˜350 mL by rotary evaporation at 28° C.and stirred at room temperature for 30 min. Heptane (700 mL) was addedand the resulting slurry was stirred at room temperature for 30 min,cooled to 4° C. and stirred for 1 h. The solids were filtered, washedwith heptane, and then dried under vacuum to yield the title compound(193.0 g, >97% purity). The filtrate was concentrated, slurried in anisopropyl acetate and heptane mixture (1:3, 60 mL) over 30 min, filteredand dried to yield additional product (45.0 g, >97% purity).

c.trans-(7-Cyano-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydronaphthalen-2-yl)-carbamicacid tert-butyl ester

Trifluoro-methanesulfonic acid7-tert-butoxycarbonylamino-8,8-diethyl-6-methoxy-5,6,7,8-tetrahydro-naphthalen-2-ylester (236.6 g, 0.49 mol) was dissolved in N,N-dimethylformamide (851mL, 10.99 mol) and water (23.8 mL, 1.32 mol) at room temperature. Thesolution was purged with nitrogen for 5 min, and then connected to housevacuum for 5 min. Nitrogen purging and exposure to vacuum was repeatedtwice. To the reaction mixture was added zinc cyanide (34.2 g, 0.29mol), tris(dibenzylideneacetone)dipalladium(0) (4.4 g, 4.8 mmol) and1,1′-bis(diphenylphosphino)ferrocene (5.4 g, 9.7 mmol) with stirring.The reaction mixture was purged with nitrogen for 5 min, heated undernitrogen at 110° C. for 1 h, cooled to room temperature and thenfiltered through celite. The filtered reaction mixture was added slowlyto water (3 L), cooled to 0° C. with stirring, stirred for 30 min at 0°C., and then filtered. The filter cake was washed with water (500 mL)and dried in air for 2 h, slurried in ethanol (1 L) with stirring over 1h, and then filtered to give the title compound (165.0 g, >96% purity).The filtrate was dried (21.6 g) and dissolved in ethanol (110 mL) withstirring over 1 h, and the resulting slurry was filtered and dried undervacuum to give additional product (10.2 g, >98% purity).

Preparation 3trans-(7-Carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydronaphthalen-2-yl)-carbamicacid tert-butyl ester

A slurry of the product of Preparation 2 (160.0 g, 446.3 mmol) andmethanol (3.3 L) was heated at 55° C. for 15 min, sodium perboratemonohydrate (280 g, 2800 mmol) and water (330 mL) was added and thereaction mixture was heated at 55° C. overnight. Additional sodiumperborate monohydrate (90 g) was added and the reaction mixture washeated at 55° C. overnight, then cooled to room temperature, and theinorganic solids were filtered off. The filtrate was transferred to a 5L flask and most of the solvent was removed by rotary evaporation. Tothe resulting slurry was added water (1.1 L) and ethyl acetate (450 mL)and the reaction mixture was stirred at room temperature for 20 min. Thereaction mixture was filtered and the filter cake was washed with water(200 mL) and then ethyl acetate (200 mL) and dried to yield the titlecompound (123 g, ˜95% purity). The filtrate was concentrated to drynessand dried under vacuum to yield additional product (18 g, 65% purity).

Preparation 4trans-7-Amino-8,8-diethyl-6-methoxy-5,6,7,8-tetrahydro-naphthalene-2-carboxylicacid amide

Acetyl chloride (278.8 mL, 3920 mmol) was added dropwise to ethanol (382mL, 6530 mmol) at −5° C. over 2 h keeping the internal temperature below20° C. The resulting solution was added portion wise over 15 min,keeping the internal temperature below 30° C., to a slurry oftrans-(7-carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydronaphthalen-2-yl)-carbamicacid tert-butyl ester (123.0 g, 327 mmol) and ethanol (500 mL) that hadbeen cooled to 10° C. The reaction mixture was stirred at roomtemperature for 2 h, and concentrated to ˜200 mL by rotary evaporation.Ethyl acetate (200 mL) was added and the resulting slurry was stirred at0° C. for 30 min, filtered and dried to yield the hydrochloride salt ofthe title compound (102 g, >98% purity) as a white solid.

Preparation 5 Carbonic acid 4-nitro-phenyl ester (R)-1-phenyl-ethylester

A mixture of (R)-1-phenyl-ethanol (60.6 g, 0.496 mol), pyridine (42.5mL, 0.526 mol) and 2-methyl-tetrahydrofuran (600 mL) was cooled to 0° C.and p-nitrophenyl chloroformate (100 g, 0.496 mol) was added over 15 minkeeping the internal temperature below 5° C. The reaction mixture waswarmed to room temperature and stirred for 2 h. To the reaction mixturewas added 1.0 M HCl in water (300 mL). Layers were separated. Theorganic layer was washed with 1N HCl (300 mL) and brine (300 mL),filtered, concentrated to dryness by rotary evaporation, and dried undervacuum to give the title compound (140 g) as a clear yellow oil.

Preparation 6(6S,7S)-7-Amino-8,8-diethyl-6-methoxy-5,6,7,8-tetrahydro-naphthalene-2-carboxylicacid amide a.((2S,3S)-7-Carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydro-naphthalen-2-yl)-carbamicacid (R)-1-phenyl-ethyl ester

A mixture of carbonic acid 4-nitro-phenyl ester (R)-1-phenyl-ethyl ester(102 g, 357 mmol), N,N-dimethylformamide (200 mL) and triethylamine(32.7 mL, 235 mmol) was stirred at room temperature overnight. To thereaction mixture was addedtrans-7-amino-8,8-diethyl-6-methoxy-5,6,7,8-tetrahydro-naphthalene-2-carboxylicacid amide hydrochloride (100 g, 320 mmol), N,N-dimethylformamide (320mL) and triethylamine (98.0 mL, 703 mmol). The reaction mixture washeated at 85° C. for 5 h and then stirred at room temperature overnight.Approximately 90% of the DMF was removed by distillation at 70° C. andthe resulting thick oil was cooled to room temperature and thenpartitioned between ethyl acetate (1.5 L) and diluted brine (500 mL).The organic layer was washed with 1M NaOH (3×500 mL) and dried withNa₂SO₄. Most of the solvent was removed by rotary evaporation, 3 volumesethyl acetate were added and resulting slurry was stirred at roomtemperature for 30 min, filtered and dried to give the title compound(48 g, >99% chemical and optical purity).

The filtrate was washed with 1M NaOH (200 mL) and then with dilutedbrine (2×200 mL). Most of the solvent was removed by rotary evaporationyielding a thick oil to which ethyl acetate (100 mL) was added. A pinchof seeds of the title compound was added and the reaction mixture wasrefrigerated at 0° C. after stirring for ˜30 min. The resulting thinslurry was stirred for 5 min and filtered; flask and filter cake werewashed with ethyl acetate (2×15 mL) to yield additional title compound(4.1 g, 97% chemical and >99% optical purity, 38% combined yield).

b.(6S,7S)-7-Amino-8,8-diethyl-6-methoxy-5,6,7,8-tetrahydronaphthalene-2-carboxylicacid amide

Acetyl chloride (193 mL, 2710 mmol) was added dropwise to ethanol (260mL, 4500 mmol) at −5° C. over 40 min keeping the internal temperaturebelow 30° C. The resulting solution was added over 5 min, at 10° C., toa mixture of((2S,3S)-7-carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydro-naphthalen-2-yl)-carbamicacid (R)-1-phenyl-ethyl ester (49.0 g, 115 mmol) and ethanol (200 mL).The reaction mixture was stirred at room temperature overnight, andconcentrated to ˜100 mL by rotary evaporation. Ethyl acetate (100 mL)was added and the resulting slurry was stirred at 0° C. for 30 min andfiltered. The filter cake was washed with ethyl acetate and dried toyield the hydrochloride salt of the title compound (30 g, >99% purity).The volume of the filtrate was reduced almost to dryness. Isopropylalcohol (20 mL) was added and the resulting thick slurry was stirred for30 min and filtered. The filter cake was washed with ethyl acetate (2×20mL) and dried under vacuum overnight to yield additional title compound(5.5 g, >97% purity). ¹H NMR (DMSO-d₆): δ (ppm) 0.49 (t, 3H), 0.63 (t,3H), 1.62 (q, 2H), 1.89 (m, 1H), 2.09 (m, 1H), 2.60 (dd, 1H), 3.22 (m,1H), 3.41 (s, 3H), 3.50 (dd, 1H), 3.82 (q, 1H), 7.19 (d, 1H), 7.31 (br,1H), 7.70 (d, 1H), 7.71 (s, 1H), 7.98 (br, 1H), 8.15 (br, 3H).

Preparation 7 Sodium(S)-4-cyclohexyl-1-hydroxy-3-methoxycarbonyl-butane-1-sulfonate a:(S)-2-Cyclohexylmethyl-4-hydroxy-butyric acid methyl ester

A mixture of (S)-2-cyclohexylmethyl-succinic acid 1-methyl ester (60.0g, 263 mmol) and tetrahydrofuran (600 mL) was stirred at roomtemperature and then cooled to −5° C. over 30 min. To the reactionmixture was added 1.0 M borane in tetrahydrofuran (520 mL) dropwise over45 min, keeping the internal temperature below 0° C. To the reactionmixture was added MeOH (100 mL) dropwise to quench the reaction. Thereaction mixture was concentrated to about 100 mL by rotary evaporation.(Trifluoromethyl)benzene (200 mL) was added and volume was reduced to 25mL by rotary evaporation. (Trifluoromethyl)benzene (100 mL) was added tothe resulting thick oil and the volume was reduced to ˜25 mL to providecrude title product (56.3 g).

b. Sodium(S)-4-cyclohexyl-1-hydroxy-3-methoxycarbonyl-butane-1-sulfonate

A mixture of (S)-2-cyclohexylmethyl-4-hydroxy-butyric acid methyl ester(44.8 g, 209 mmol) and DCM (310 mL) was cooled to 5° C. with stirring.To the reaction mixture was added a solution of potassium bromide (2.5g, 21 mmol) and sodium bicarbonate (2.4 g, 29 mmol) in distilled water(130 mL), and then 2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO) (0.33 g,2.1 mmol), followed by the addition of sodium hypochlorite (140 mL, 210mmol) at the rate of 130 mL/h keeping the internal temperature in therange of 6-8° C. The reaction mixture was stirred for 15 min and DCM(200 mL) was added. Layers were separated and the organic layer waswashed with saturated brine (200 mL), and dried with Na₂SO₄.

To the organic layer was added EtOAc (40 mL) followed by the addition ofsodium bisulfite (21.8 g, 209 mmol). The reaction solution wasconcentrated to remove half of the DCM (˜175 mL) by rotary evaporation.Water (2 mL) were added to the reaction solution which was stirred atroom temperature overnight. The resulting slurry was filtered; thefilter cake was dried under vacuum overnight to yield the title compound(61.9 g). ¹H NMR (DMSO-d₆): δ (ppm) 0.78 (m, 2H), 0.95-1.20 (m, 4H),1.33 (m, 1H), 1.40-1.95 (m, 5H), 2.45-2.65 (m, 1H), 3.21 (m, 2H), 3.45(s, 3H), 3.6-3.8 (m, 1H), 5.18 (d, 1H).

Preparation 8 Sodium(S)-3-benzyloxycarbonyl-4-cyclohexyl-1-hydroxy-butane-1-sulfonate a.(S)-2-cyclohexylmethyl-4,4-dimethoxy-butyric acid methyl ester

To a slurry of sodium(S)-4-cyclohexyl-1-hydroxy-3-methoxycarbonyl-butane-1-sulfonate (400.0g, 1.26 mol) and methanol (2 L) was added 4.0 M HCl in 1,4-dioxane (400mL) and the reaction mixture was stirred for 15 min. Trimethoxymethane(340 mL, 3.11 mol) was added and reaction mixture was heated at 50° C.overnight, and then cooled to room temperature. White solids werefiltered off and discarded. Most of the solvent was removed from thefiltrate by rotary evaporation. Ethyl acetate (800 mL) was addedresulting in more precipitation. The white precipitate was removed byfiltration. Solvent was removed from the filtrate by rotary evaporationand then under high vacuum at room temperature overnight to yield thetitle compound (211 g) as a thick oil. ¹H NMR (400 MHz, DMSO-d₆): δ(ppm) 4.25 (t, 1H), 3.57 (s, 3H), 3.18 (s, 6H), 2.43 (m, 1H), 1.55-1.81(m, 2H), 1.50-1.72 (m, 5H), 1.20-1.48 (m, 2H), 1.05-1.21 (m, 4H),0.71-0.92 (m, 2H).

b. Potassium (S)-2-cyclohexylmethyl-4,4-dimethoxy-butyrate

Potassium hydroxide (289.6 g, 2322 mmol) was added to a solution of theproduct of the previous step (200.0 g, 0.77 mol) in methanol (700 mL) inone portion and the reaction mixture was stirred at RT for 20 h.Hydrogen chloride (130 mL, 1.5 mol) was added slowly until the reactionmixture had a pH ˜8 (color change from greenish to orange) resulting inprecipitation of fine solids. Solids were removed by filtration. Solventwas removed from the filtrate. Acetonitrile (1 L) was added to the crudeproduct and the resulting slurry was stirred at room temperatureovernight. The thick slurry was filtered, the filter cake was washedwith acetonitrile (50 mL) and dried to yield a first crop of the titlecompound (133 g) as an off-white solid. Solvent was removed from thefiltrate which was then dried under vacuum to yield about 100 g of apasty solid. MTBE (500 mL) was added and the solids were stirred at RTovernight resulting in a thick slurry which was filtered and dried underhigh vacuum to yield a second crop of the title compound (82 g). ¹H NMR(400 MHz, DMSO-d₆): δ (ppm) 4.28 (dd, 1H), 3.12 (s, 3H), 3.15 (s, 3H),1.95 (m, 1H), 1.75 (m, 1H), 1.51-1.65 (m, 6H), 1.22-1.39 (m, 2H),1.05-1.20 (m, 4H), 0.85-0.93 (m, 1H), 0.65-0.81 (m, 2H).

c: (S)-2-Cyclohexylmethyl-4,4-dimethoxy-butyric acid benzyl ester

Benzyl bromide (50.54 mL, 424.9 mmol) was added to a slurry of theproduct of the previous step (150.0 g, 531.1 mmol) in acetonitrile (2.0L) in one portion and the heterogeneous reaction mixture was stirred atroom temperature overnight. Additional benzyl bromide (5.05 mL, 42.49mmol) was added and the reaction mixture was stirred at room temperaturefor 18 h. Solids were removed by filtration. The filtrate was dried byrotary evaporation and then under high vacuum overnight yielding thetitle compound (162 g). ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 7.22-7.40 (m,5H), 5.0-5.15 (q, 2H), 4.23 (t, 1H), 3.15 (s, 3H), 3.17 (s, 3H), 2.52(m, 1H), 1.78 (m, 1H), 1.69 (m, 1H), 1.45-1.61 (m, 6H), 1.20-1.43 (m,2H), 1.0-1.15 (m, 4H), 0.70-0.83 (m, 2H).

d. Sodium(S)-3-benzyloxycarbonyl-4-cyclohexyl-1-hydroxy-butane-1-sulfonate

To a mixture of the product of the previous step (160.0 g, 478.4 mmol)and acetonitrile (1.0 L) was added 1.0 M HCl in water (1.2 L) and thereaction mixture was heated at 35-40° C. for 2 h. Ethyl acetate (1.2 L)was added, phases were separated, and the organic layer was washed withbrine (1 L). Sodium bisulfite (74.7 g, 718 mmol) was added to the wetorganic layer and the reaction mixture was stirred at RT overnight. Mostof the solvent was removed by rotary evaporation and acetonitrile (1 L)was added and the resulting slurry was stirred at RT overnight. Theresulting thick white slurry was filtered, the filter cake was washedwith acetonitrile (2×100 mL) and dried under vacuum to yield the titlecompound (200 g, >98% purity) as a white solid. ¹H NMR (400 MHz,DMSO-d₆): δ (ppm) 7.23-7.41 (m, 5H), 5.30 (d, 1H), 4.98-5.18 (q, 2H),3.75-3.88 (m, 1H), 3.60-3.79 (m, 1H), 2.05 (m, 0.5H), 1.45-1.82 (m,2.5H), 1.45-1.60 (m, 5H), 1.20-1.42 (m, 2H), 1.0-1.17 (m, 4H), 0.69-0.82(m, 2H).

Example 1(S)-4-((2S,3S)-7-Carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydronaphthalen-2-ylamino)-2-cyclohexylmethyl-butyricacid a.(S)-4-((2S,3S)-7-Carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydronaphthalen-2-ylamino)-2-cyclohexylmethyl-butyricacid methyl ester

To a slurry of sodium(S)-4-cyclohexyl-1-hydroxy-3-methoxycarbonyl-butane-1-sulfonate (25.8 g,81.5 mmol) and 2-methyl-tetrahydro-furan (300 mL) was added 1.0 M NaOHin water (76.1 mL) and the reaction mixture was stirred for 20 min atRT. To the reaction mixture was added(6S,7S)-7-amino-8,8-diethyl-6-methoxy-5,6,7,8-tetrahydro-naphthalene-2-carboxylicacid amide hydrochloride (17.0 g, 54.3 mmol); the reaction mixture wasstirred for 40 min at RT, sodium triacetoxyborohydride (46.1 g, 217mmol) was added in 4 portions. The reaction mixture was stirred at RTovernight after the first two portions. Water (200 mL) and MeTHF (100mL) were added; the phases were separated and the organic layer waswashed with 1 M NaOH (2×200 mL), diluted brine (200 mL) dried withNa₂SO₄ and solvent was removed to yield crude title intermediate (22 g)as a glassy yellow solid.

Crude product was purified by reverse-phase chromatography using aMicrosorb 100-10 BDS 4 inch column. Crude product was dissolved in 1:1acetonitrile: 1 M aq. HCl (150 mL) solvent mixture and eluted with water(0.1% HCl)/acetonitrile mobile phase (10-40% gradient). Pure fractions(>98%) were combined, most of the acetonitrile was removed by rotaryevaporation, pH was adjusted to pH ˜12 with solid Na₂CO₃ and purifiedproduct was extracted with MeTHF (3×1 L). Combined organic layers weredried with Na₂SO₄ and solvent removed to yield the title compound (16.5g)

b.(S)-4-((2S,3S)-7-Carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydronaphthalen-2-ylamino)-2-cyclohexylmethyl-butyricacid

To a solution of(S)-4-((2S,3S)-7-carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydro-naphthalen-2-ylamino)-2-cyclohexylmethyl-butyricacid methyl ester (12.0 g, 25.4 mmol) in methanol was added 5.0 M NaOH(25 mL) and the reaction mixture was heated at 30° C. for 8 h and thenat 25° C. overnight. Most of the methanol solvent was removed by rotaryevaporation at 25° C., water (100 mL) and isopropyl acetate (100 mL) wasadded and the resulting mixture was stirred for 15 min. The bottom twoof three layers were extracted with isopropyl acetate (100 mL). Bottomlayers were cooled to −5° C. and MeTHF (200 mL) was added and thenconcentrated HCl (˜15 mL) was added in portions until pH ˜2. Phases wereseparated, water layer was washed with MeTHF (100 mL) and combinedorganic layers were dried with Na₂SO₄. Most of the organic solvent wasremoved by rotary evaporation, ethyl acetate (200 mL) was added and thevolume was reduced to 50 mL. Ethyl acetate (200 mL) was added and theresulting slurry was stirred/triturated at RT for 3 h. Product wasfiltered under nitrogen and dried under vacuum for 48 h to yield thehydrochloride salt of the title compound (11 g, 98.2% purity) as a whitesolid. ¹H NMR (DMSO-d₆): δ (ppm) 0.54 (t, 3H), 0.63 (t, 3H), 0.82 (m,2H), 1.05-1.3 (m, 6H), 1.45 (m, 1H), 1.55-2.0 (m, 10H), 2.40 (m, 1H),2.67 (dd, 1H), 3.06 (m, 1H), 3.22 (m, 1H), 3.30 (dd, 1H), 3.41 (s, 3H),3.45 (dd, 1H), 4.05 (m, 1H), 7.19 (d, 1H), 7.50 (br, 1H), 7.69 (d, 1h),7.70 (s, 1H), 7.95 (br, 2H), 9.26 (br, 1H).

Example 2(S)-4-((2S,3S)-7-Carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydronaphthalen-2-ylamino)-2-cyclohexylmethyl-butyricacid

(S)-4-((2S,3S)-7-Carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydronaphthalen-2-ylamino)-2-cyclohexylmethyl-butyricacid hydrochloride (2.18 g, 4.40 mmol) was dissolved in methanol (30 mL)and water (30 mL) and 1.0 M NaOH (4.65 mL) was added. Methanol wasremoved by rotary evaporation resulting in precipitation and 1.0 M HCl(0.045 mL) was added resulting in additional precipitation. The solidswere extracted with DCM: isopropyl alcohol (4:1, 3×40 mL) and dried oversodium sulfate. Water (30 mL) was added and the organics were removed byrotary evaporation providing a gummy precipitate in water. Acetonitrile(25 mL) was added and the reaction mixture was lyophilized to providethe title compound as an amorphous solid. (1.99 g).

Example 3 Crystalline(S)-4-((2S,3S)-7-Carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydronaphthalen-2-ylamino)-2-cyclohexylmethyl-butyricacid a.(S)-4-((2S,3S)-7-Carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydronaphthalen-2-ylamino)-2-cyclohexylmethyl-butyricacid benzyl ester hydrochloride

To a suspension of sodium(S)-3-benzyloxycarbonyl-4-cyclohexyl-1-hydroxy-butane-1-sulfonate (160g, 400 mmol), the product of Preparation 8, in MeTHF (2.0 L) and water(600 mL) was added 1.0 M NaOH in water (400 mL) and the reaction mixturewas stirred at room temperature for 90 min. Phases were separated andthe solution was concentrated to a volume of ˜300 mL.

The resulting concentrated solution was added to a slurry of(6S,7S)-7-amino-8,8-diethyl-6-methoxy-5,6,7,8-tetrahydro-naphthalene-2-carboxylicacid amide hydrochloride (100.0 g, 319.7 mmol) in DMF (1 L). Resultingslurry was stirred at room temperature for 2 h, the reaction mixture wascooled to 0° C. followed by portion-wise addition of sodiumtriacetoxyborohydride (169 g, 799 mmol) over 15 min. The reactionmixture was stirred at RT overnight, cooled to 10° C. and then 1.0 MNaOH in water (3 L) and ethyl acetate (5 L) were added. The reactionmixture was stirred for 10 min, phases were separated, and the organiclayer was washed with diluted brine (1:1, 2 L). To the organic layer wasadded 1.0 M HCl in water (520 mL, 520 mmol) and most of the ethylacetate was removed by rotary evaporation. Water (500 mL) and ethanol (1L) were added and the volume was slowly reduced by rotary evaporation to˜1 L. The resulting off-white free-flowing slurry was stirred at RTovernight. Product was isolated by filtration, flask and filter cakewere washed with water (2×200 mL) and then dried to yield the titlecompound (175 g) as a white solid (˜99% purity, 90% yield based onaminotetralin reagent). ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 9.33 (br,1H), 8.09 (br, 1H), 7.98 (s, 1H), 7.70 (s, 1H), 7.68 (d, 1H), 7.28-7.36(m, 2H), 7.19 (d, 1H), 5.10 (q, 2H), 4.04 (m, 1H), 3.45 (dd, 1H), 3.38(s, 3H), 3.25 (m, 2H), 3.05 (m, 1H), 2.62 (m, 2H), 1.95-2.15 (m, 2H),1.61-1.82 (m, 3H), 1.50-1.61 (m, 4H), 1.42-1.50 (m, 1H), 1.24-1.32 (m,1H), 0.98-1.18 (m, 4H), 0.71-0.89 (m, 2H), 0.63 (t, 3H), 0.52 (t, 3H)

b. Crystalline(S)-4-((2S,3S)-7-Carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydronaphthalen-2-ylamino)-2-cyclohexylmethyl-butyricacid

The product of the previous step (175.0 g, 299 mmol) was partitionedbetween ethyl acetate (2.5 L), water (1 L) and 1.0 M NaOH in water (300mL, 299 mmol). Phases were separated, the organic layer was washed withdiluted brine (1:1, 250 mL), and dried with sodium sulfate. Solvent wasremoved by rotary evaporation and the resulting product dried overnightunder high vacuum to provide the free-base intermediate(S)-4-((2S,3S)-7-carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydro-naphthalen-2-ylamino)-2-cyclohexylmethyl-butyricacid benzyl ester (˜160 g) as a sticky solid.

The free-base intermediate was dissolved in a mixture of acetonitrile(1.6 L) and water (300 mL). To half of the solution (1 L) was added 10%palladium (10 g, 9 mmol) on carbon (wet). The reaction mixture waspurged with nitrogen and then with hydrogen for 2 min and then exposedto 10-15 psi H₂ for 3 h at RT. The reaction mixture was filtered throughcelite, and the flask and filter cake were washed with acetonitrile (50mL). The yellowish filtrate was stirred with thiol-modified silica (10g) at RT for 2 h and then filtered through celite. Most of the solventwas removed by rotary evaporation at 25° C. Acetonitrile (500 mL) wasadded and most of the solvent was removed by rotary evaporation.Additional acetonitrile (500 mL) was added resulting in fastprecipitation of sticky solids. The reaction mixture was stirredvigorously at room temperature overnight resulting in a free-flowingoff-white slurry. Product was isolated by filtration; the filter cakewas washed with acetonitrile (2×50 mL) and then dried under vacuum toyield the crystalline title compound (56 g, 98.8% purity). Water content0.49% (w/w). ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 7.89 (br, 1H), 7.65 (s,1H), 7.60 (d, 1H), 7.22 (br, 1H), 7.11 (d, 1H), 3.55 (m, 1H), 3.38 (s,3H), 3.25 (dd, 1H), 2.95 (m, 1H), 2.59 (d, 1H), 2.49 (m, 2H), 1.81 (m,2H), 1.49-1.63 (m, 5H), 1.41-1.50 (m, 2H), 1.05-1.25 (m, 4H), 0.72-0.90(m, 2H), 0.45 (t, 3H), 0.57 (t, 3H).

Note: In the text of the following examples(S)-4-((2S,3S)-7-Carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydronaphthalen-2-ylamino)-2-cyclohexylmethyl-butyricacid is referenced as compound 1.

Example 4 Crystallization of(S)-4-((2S,3S)-7-Carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydronaphthalen2-ylamino)-2-cyclohexylmethyl-butyricacid (Form I)

Amorphous compound 1 (100 mg, 0.22 mmol) was dissolved in isopropanol(0.83 mL). After four days, crystals were observed in the solution. Themother liquor was decanted and 1:1 acetonitrile:ethyl acetate (0.2 mLand then an additional 0.3 mL) was added to provide a slurry of thetitle compound.

Example 5 Crystallization of(S)-4-((2S,3S)-7-Carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydronaphthalen-2-ylamino)-2-cyclohexylmethyl-butyricacid (Form I)

Amorphous compound 1 (33 mg, 0.072 mmol) was dissolved in a premixedsolution of acetonitrile (0.04 mL), 1-propanol (0.0605 mL), and water(0.0035 mL) and the process mixture was stirred at RT overnight. Aprecipitate started to form in less than about 1 h. The solids werecollected by filtration, washed with 3:2 acetonitrile:isopropanol (2 mL)and dried under vacuum to provide the title compound.

Example 6 Crystallization of(S)-4-((2S,3S)-7-Carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydronaphthalen-2-ylamino)-2-cyclohexylmethyl-butyricacid (Form I)

Amorphous compound 1 (84.2 mg, 0.18 mmol) was dissolved in a mixture ofwater (0.003 mL) and methanol (0.027 mL) and then a mixture of water(0.013 mL) and acetonitrile (0.090 mL) was added. Crystals were observedwithin 2 min. The process mixture was held for 3 days at RT withoutstirring. The mother liquor was decanted and the solids were isolated byvacuum filtration and washed with acetonitrile to provide the titlecompound (54 mg, 64% yield).

Example 7 Crystallization of(S)-4-((2S,3S)-7-Carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydronaphthalen-2-ylamino)-2-cyclohexylmethyl-butyricacid (Form II)

Amorphous compound 1 (70 mg, 0.15 mmol) was dissolved in a premixedsolution of acetonitrile (0.136 mL) and isopropanol (0.090 mL) and theprocess mixture was stirred at RT overnight. A precipitate started toform in less than about 1 h. The solids were collected by filtration,washed with 3:2 acetonitrile:isopropanol (2 mL) and dried under vacuumto provide the title compound (64 mg).

Example 8 Crystallization of(S)-4-((2S,3S)-7-Carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydronaphthalen-2-ylamino)-2-cyclohexylmethyl-butyricacid hydrochloride

Amorphous hydrochloride salt of compound 1 (56.7 mg, 0.12 mmol) wasdispersed in diethylene glycol dimethylether (DEGDME) (0.50 mL), heatedto 50° C. for 20 min and slowly cooled overnight. The process mixturewas left at ambient temperature for 11 days. Resulting crystals wereisolated and washed with DEGDME (˜0.1 mL) to provide the title compound.

Example 9 Crystallization of(S)-4-((2S,3S)-7-Carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydronaphthalen-2-ylamino)-2-cyclohexylmethyl-butyricacid hydrochloride

Amorphous hydrochloride salt of compound 1 (72.2 mg, 0.15 mmol) wasdispersed in ethyl acetate (1.0 mL), sonicated for less than 1 min,heated to 50° C. and slowly cooled. The process mixture was left atambient temperature for 3 days. Resulting crystals were isolated toprovide the title compound.

Example 10 Crystallization of(S)-4-((2S,3S)-7-Carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydronaphthalen-2-ylamino)-2-cyclohexylmethyl-butyricacid hydrochloride

Amorphous hydrochloride salt of compound 1 (50 mg, 0.11 mmol) wasdispersed in ethyl acetate (0.5 mL) and stirred at RT for 4 days. Theresulting crystals were isolated to provide the title compound (30 mg).

Examples 11-15 Properties of Solid Forms of the Invention

Samples of crystalline(S)-4-((2S,3S)-7-carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydro-naphthalen-2-ylamino)-2-cyclohexylmethyl-butyricacid (compound 1) prepared in Examples 3 and 7 and of the crystallinehydrochloride salt of compound 1, prepared in Example 9, as well as asample of crystalline(S)-4-((2S,3S)-7-Carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydro-naphthalen-2-ylamino)-2-cyclohexylmethyl-butyricacid benzyl ester hydrochloride, (hydrochloride salt of compound 2),prepared as in Example 3a were analyzed by x-ray powder diffraction(XRPD), differential scanning calorimetry (DSC), and thermogravimetricanalysis (TGA).

Example 11 X-Ray Powder Diffraction

X-ray powder diffraction patterns of FIGS. 1, 4, and 6 were obtainedwith a Rigaku diffractometer using Cu Kα (30.0 kV, 15.0 mA) radiation.The analysis was performed with the goniometer running incontinuous-scan mode of 2° per min (FIG. 1) or 3° per min (FIGS. 4, 6,and 8) with a step size of 0.03° over a range of 2 to 40°. Samples wereprepared on quartz specimen holders as a thin layer of powderedmaterial. The instrument was calibrated with a silicon standard. FIG. 8was obtained with a Thermo XTRA model ARL diffractometer scanning at1.22° per min with a step size of 0.03° over a range of 2 to 40°.

Example 12 Thermal Analysis

Differential scanning calorimetry (DSC) was performed using a TAInstruments Model Q-100 module. Data were collected and analyzed usingTA Instruments Thermal Advantage for Q Series™ software. A sample ofabout 1-10 mg was accurately weighed into an aluminum pan with lid. Thesample was evaluated using a linear heating ramp of 10° C./min from 5°C. to, typically, 265° C. The DSC cell was purged with dry nitrogenduring use. Representative DSC traces for samples of crystalline Form Iand Form II, of the crystalline hydrochloride salt of compound 1, aswell as the crystalline hydrochloride salt of compound 2, are shown inFIGS. 2, 5, 7, and 9 respectively.

Thermogravimetric analysis (TGA) was performed using a TA InstrumentsModel Q-500 module. Data were collected and analyzed using TAInstruments Thermal Advantage for Q Series™ software. A sample weighingabout 1-5 mg was placed in an aluminum pan on a platinum cradle andscanned from ambient temperature to 300° C. with a linear heating rateof 10° C./min. The balance and furnace chambers were purged withnitrogen during use. Representative TGA traces for samples ofcrystalline Form I and of a crystalline hydrochloride salt of compound 1are also shown in FIGS. 2 and 7, respectively.

Example 13 Dynamic Moisture Sorption Assessment

Dynamic moisture sorption (DMS) assessment was performed at 25° C. usinga VTI atmospheric microbalance, SGA-100 system (VTI Corp., Hialeah, Fla.33016). A sample size of approximately 5-10 mg was used and the humiditywas set at the ambient value at the start of the analysis. A typical DMSanalysis consisted of three or four scans: ambient to 2% relativehumidity (RH), 2% RH to 90% RH, 90% RH to 5% RH at a scan rate of 5%RH/step, and, in some case a second adsorption 2% RH to 90% RH. The masswas measured every two minutes and the RH was changed to the next value(±5% RH) when the mass of the sample was stable to within 0.02% for 5consecutive points. A representative DMS trace for a sample ofcrystalline Form 1 prepared in Example 3 is shown in FIG. 3.

Example 14 X-Ray Diffraction Crystal Structure Analysis

A needle-like crystal of crystalline Form I having dimensions of0.20×0.10×0.06 mm, prepared in Example 6, was analyzed. X-raydiffraction crystal structure data was obtained using a Nonius Kappa-CCDdiffractometer using Mo K_(α) radiation. Full sphere data was collectedup to θ=27.5 degrees at a temperature of 120° K and was analyzed usingSHELX-97 software. The following lattice parameters were derived: unitcell is orthorhombic with dimensions a=7.546 Å, b=17.003 Å, c=20.628 Å,cell volume (V) of 2646.7 Å³; calculated density is 1.151 g/cm³; spacegroup is P2₁2₁2₁. Powder x-ray diffraction peaks predicted from thederived atomic positions were judged by visual inspection to be inexcellent agreement with the experimentally determined peak positions.

Assay 1: Radioligand Binding Assay on Human Mu, Human Delta and GuineaPig Kappa Opioid Receptors

a. Membrane Preparation

CHO-K1 (Chinese Hamster Ovary) cells stably transfected with human muopioid or with guinea pig kappa receptor cDNA were grown in mediumconsisting of Ham's-F12 media supplemented with 10% FBS, 100 units/mlpenicillin-100 μg/mL streptomycin and 800 μg/mL Geneticin in a 5% CO₂,humidified incubator @ 37° C. Receptor expression levels (B_(max)˜2.0and ˜0.414 pmol/mg protein, respectively) were determined using[³H]-Diprenorphine (specific activity ˜50-55 Ci/mmol) in a membraneradioligand binding assay.

Cells were grown to 80-95% confluency (<25 subculture passages). Forcell line passaging, the cell monolayer was incubated for 5 minutes atroom temperature and harvested by mechanical agitation in 10 mL of PBSsupplemented with 5 mM EDTA. Following resuspension, cells weretransferred to 40 mL fresh growth media for centrifugation for 5 minutesat 1000 rpm and resuspended in fresh growth medium at the appropriatesplit ratio.

For membrane preparation, cells were harvested by gentle mechanicalagitation with 5 mM EDTA in PBS followed by centrifugation (2500 g for 5minutes). The pellets were resuspended in Assay Buffer (50 mM4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acidN-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES)), pH7.4, and homogenized with a polytron disrupter on ice. The resultanthomogenates were centrifuged (1200 g for 5 minutes), the pelletsdiscarded and the supernatant centrifuged (40,000 g for 20 minutes). Thepellets were washed once by resuspension in Assay Buffer, followed by anadditional centrifugation (40,000 g for 20 minutes). The final pelletswere resuspended in Assay Buffer (equivalent 1 T-225 flask/1 mL assaybuffer). Protein concentration was determined using a Bio-Rad BradfordProtein Assay kit and membranes were stored in frozen aliquots at −80°C., until required.

Human delta opioid receptor (hDOP) membranes were purchased from PerkinElmer. The reported K_(d) and B_(max) for these membranes determined bysaturation analyses in a [³H]-Natrindolc radioligand binding assays were0.14 nM (pK_(d)=9.85) and 2.2 pmol/mg protein, respectively. Proteinconcentration was determined using a Bio-Rad Bradford Protein Assay kit.Membranes were stored in frozen aliquots at −80° C., until required.

b. Radioligand Binding Assays

Radioligand binding assays were performed in an Axygen 1.1 mL deep well96-well polypropylene assay plate in a total assay volume of 200 μLcontaining the appropriate amount of membrane protein (˜3, ˜2 and ˜20 μgfor mu, delta and kappa, respectively) in Assay Buffer, supplementedwith 0.025% bovine scrum albumin (BSA). Saturation binding studies fordetermination of K_(d) values of the radioligand were performed using[³H]-Diprenorphine at 8-12 different concentrations ranging from 0.001nM-5 nM. Displacement assays for determination of pKi values ofcompounds were performed with [³H]-Diprenorphine at 0.5, 1.2, and 0.7 nMfor mu, delta, and kappa, respectively, and eleven concentrations ofcompound ranging from 10 μM-100

Binding data were analyzed by nonlinear regression analysis with theGraphPad Prism Software package (GraphPad Software, Inc., San Diego,Calif.) using the 3-parameter model for one-site competition. The curveminimum was fixed to the value for nonspecific binding, as determined inthe presence of 10 μM naloxone. K, values for test compounds werecalculated, in Prism, from the best fit IC₅₀ values, and the K_(d) valueof the radioligand, using the Cheng-Prusoff equation(K_(i)=IC₅₀/(1+([L]/K_(d))) where [L]=the concentration of[³H]-Diprenorphine Results are expressed as the negative decadiclogarithm of the K_(i) values, pK_(i).

Test compounds having a higher pK_(i) value in these assays have ahigher binding affinity for the mu, delta, or kappa opioid receptor.Compound 1 exhibited a pK_(a) value of 9.4 at the human mu opioidreceptor.

Assay 2: Agonist Mediated Activation of the Mu-Opioid Receptor inMembranes Prepared from CHO-K1 Cells Expressing the Human Mu-OpioidReceptor

In this assay, the potency and intrinsic activity values of testcompounds were determined by measuring the amount of bound GTP-Eupresent following receptor activation in membranes prepared from CHO-K1cells expressing the human mu opioid receptor.

a. Mu Opioid Receptor Membrane Preparation:

Human mu opioid receptor (hMOP) membranes were either prepared asdescribed above or were purchased from Perkin Elmer. The reported pK_(d)and B_(max) for the purchased membranes determined by saturationanalyses in a [³H]-Diprenorphine radioligand binding assays was 10.06and 2.4 pmol/mg protein, respectively. Protein concentration wasdetermined using a Bio-Rad Bradford Protein Assay kit. Membranes werestored in frozen aliquots at −80° C., until required. Lyophilized GTP-Euand GDP were diluted to 10 μM and 2 mM, respectively, in doubledistilled H₂O then mixed and permitted to sit at room temperature for 30minutes prior to transfer to individual aliquots samples for storage at−20° C.

b. Human Mu GTP-Eu Nucleotide Exchange Assay

GTP-Eu nucleotide exchange assays were performed using the DELPHTAGTP-binding kit (Perkin/Elmer) in AcroWell 96 well filter platesaccording to the manufacturer's specifications. Membranes were preparedas described above, and prior to the start of the assay, aliquots werediluted to a concentration of 200 μg/mL in Assay Buffer (50 mM HEPES, pH7.4 at 25° C.), then homogenized for 10 seconds using a Polytronhomogenizer. Test compounds were received as 10 mM stock solutions inDMSO, diluted to 400 μM into Assay Buffer containing 0.1% BSA, andserial (1:5) dilutions then made to generate ten concentrations ofcompound ranging from 40 μM 80 μM-GDP and GTP-Eu were diluted to 4 μMand 40 nM, respectively, in Assay Buffer. The assay was performed in atotal volume of 100 μL containing 5 μg of membrane protein, testcompound ranging from 10 pM-20 μM), 1 μM GDP, and 10 nM GTP-Eu dilutedin 10 mM MgCl₂, 50 mM NaCl, and 0.0125% BSA, (final assayconcentrations). A DAMGO (Tyr-D-Ala-Gly-(methyl)Phe-Gly-ol)concentration-response curve (ranging from 12.8 pM-1 μM) was included onevery plate.

Assay plates were prepared immediately prior to assay following theaddition of 25 μL of Assay Buffer, 25 μL of test compound, and 25 μL GDPand GTP-Eu. The assay was initiated by the addition of 25 μL membraneprotein and allowed to incubate for 30 minutes. The assay plates werethen filtered with a Waters vacuum manifold connected to the housevacuum regulated to 10-12 in. Hg and washed with room temperature GTPWash Solution (2×300 mL). The bottoms of the plates were blotted toremove excess liquid. The plates were then immediately read to determinethe amount of bound GTP-Eu by measuring Time Resolved Fluorescence (TRF)on a Packard Fusion Plate ReaderVehicle: DMSO not to exceed 1% finalassay concentration.

The amount of bound GTP-Eu is proportional to the degree of activationof the mu opioid receptors by the test compound. The intrinsic activity(IA), expressed as a percentage, was determined as the ratio of theamount of bound GTP-Eu observed for activation by the test compound tothe amount observed for activation by DAMGO which is presumed to be afull agonist (IA=100). Compound 1 demonstrated an intrinsic activity of−8 in this assay. Thus, the present active agent has been shown to be anantagonist.

Assay 3: Rat Model of In Vivo Efficacy

In this assay the efficacy of test compounds was evaluated in a model ofgastrointestinal transit, which evaluates peripheral activity. Thisstudy was approved by the Institutional Animal Care and Use Committee atTheravance, Inc. and conformed to the Guide for the Care and Use ofLaboratory Animals published by the National Academy of Sciences(©1996).

a. Rat Gastric Emptying Assay

Test compounds were evaluated in the rat gastric emptying assay todetermine their ability to reverse loperamide-induced delayed gastricemptying. Rats were fasted up overnight prior to administration of testcompounds or vehicle by intravenous, subcutaneous, intramuscular or oralroutes of administration at doses ranging from 0.001 to about 30milligrams/kilogram (mg/kg). The administration of test compound wasfollowed by subcutaneous administration of loperamide at a dose of 1mg/kg or vehicle. Five minutes post loperamide or vehicleadministration, a non-nutritive, non-absorbable charcoal meal wasadministered via oral gavage and animals were allowed free access towater for the sixty minute duration of the experiment. Animals were theneuthanized via carbon dioxide asphyxiation followed by thoracotomy andthe stomach was carefully excised. The stomach was ligated at the loweresophageal sphincter and the pyloric sphincter to prevent additionalemptying during tissue removal. Gastric weight was then determined afterremoval of the ligatures.

b. Data Analysis and Results

Data was analyzed using the GraphPad Prism Software package (GraphPadSoftware, Inc., San Diego, Calif.). Percent reversal curves wereconstructed by non-linear regression analysis using the sigmoidal doseresponse (variable slope) model and best-fit ID₅₀ values werecalculated. Curve minima and maxima were fixed to loperamide controlvalues (indicating 0% reversal) and vehicle controls (indicating 100%reversal), respectively. Results are expressed as ID₅₀, the doserequired for 50% reversal of the effects of loperamide, in milligramsper kilogram. Compound 1, administered orally, exhibited an ID₅₀ valueof 0.09 mg/kg in the gastric emptying model.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto. Additionally, all publications, patents, andpatent documents cited hereinabove are incorporated by reference hereinin full, as though individually incorporated by reference.

What is claimed is:
 1. A process for preparing crystalline(S)-4-((2S,3S)-7-carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydronaphthalen-2-ylamino)-2-cyclohexylmethyl-butyricacid (compound 1), the process comprising deprotecting(S)-4-((2S,3S)-7-carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydronaphthalen-2-ylamino)-2-cyclohexylmethyl-butyricacid benzyl ester by catalytic hydrogenolysis in the presence of a polardiluent comprising between about 10% and about 20% water to formcrystalline compound
 1. 2. A process for preparing crystalline(S)-4-((2S,3S)-7-carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydronaphthalen-2-ylamino)-2-cyclohexylmethyl-butyricacid (compound 1), the process comprising: (a) reacting(S)-2-cyclohexylmethyl-4-oxo-butyric acid benzyl ester (4)

with(6S,7S)-7-amino-8,8-diethyl-6-methoxy-5,6,7,8-tetrahydronaphthalene-2-carboxylicacid amide (3)

to provide(S)-4-((2S,3S)-7-carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydro-naphthalen-2-ylamino)-2-cyclohexylmethyl-butyricacid benzyl ester (2)

(b) deprotecting the compound of formula 2 by catalytic hydrogenolysisin the presence of a polar diluent comprising between about 10% andabout 20% water to provide crystalline compound 1.